GCAMP6 mice for determination of mechanisms of chronic muscle ache, pain and fatigue. Muscle pain and muscle fatigue are symptoms in many chronic diseases including Fibromyalgia, Chronic Fatigue Syndrome, Myofacial Pain, Chronic Tension Headache, and Temporomandibular Disorder. Many more patients have degraded quality of life because of short term myalgia and fatigue that sometimes remits with treatment, or for unknown reasons, becomes chronic. Our long term goal is to determine the fundamental mechanisms that signal intense muscle pain, ache and fatigue to sensory and motor systems. We have previously used discoveries in mouse models to prove that combinations of protons, lactate, and ATP are necessary and sufficient to activate muscle sensory neurons. In translational studies in human subjects we showed that combination of these three metabolites activated the sensations of muscle ache and fatigue in human subjects. Here we propose to create a transgenic mouse that will make it possible, for the first time, to image the activity of sensory neurons that signal pain and fatigue in functioning skeletal muscle. This will allow us to establish the molecular and cellular mechanisms of the sensory signaling pathways for cognitive sensations of muscle pain and muscle fatigue. It will also make it possible to directly observe the mechanisms of several controversial phenomena in muscle pain including trigger points, the pulsating nature of muscle ache and chronic and migraine headache, lymphatic disease association with muscle fatigue and muscle pain, and sympathetic activation enhancement of muscle pain. The specific aims for this proposal are: 1) Create mice that have endogenous calcium sensors with promoters that allow expression in Group III/IV muscle innervating sensory neurons. 2) Use these mice to record III/IV afferents responses to low and high metabolites using anesthetized in vivo preparations. This will allow comparisons with the digit muscle recordings collected from teased muscle nerve fibers previously, and dorsal root ganglion neurons recorded with calcium imaging. With this method we will also determine the location and structures that physiologically identified Group III/IV receptor endings innervate (both nociceptors and ergoreceptors of all types e.g., mechanoreceptor and metaboreceptor).